Synthetic Surfaces For Culturing Stem Cell Derived Oligodendrocyte Progenitor Cells

ABSTRACT

Synthetic surfaces suitable for culturing stem cell derived oligodendrocyte progenitor cells contain acrylate polymers formed from one or more acrylate monomers. The acrylate surfaces, in many cases, are suitable for culturing stem cell derived oligodendrocyte progenitor cells in chemically defined media.

PRIORITY

This application claims priority to U.S. provisional Appl. No.61/063,010, filed Jan. 30, 2008, and is incorporated by reference in itsentirety.

FIELD

The present disclosure relates to cell culture articles and methods ofuse thereof, and more particularly to articles for supporting theculture of stem cell derived oligodendrocyte progenitor cells.

BACKGROUND

Pluripotent stem cells, such as human embryonic stem cells (hESCs) havethe ability to differentiate into any of the three germ layers, givingrise to any adult cell type in the human body. This unique propertyprovides a potential for developing new treatments for a number ofserious cell degenerative diseases, such as diabetes, spinal cordinjury, heart disease and the like. For example, spinal cord damage isgenerally irreversible with current treatments, leaving approximately250,000 Americans in a devastating position. However, as stem cellresearch has developed, new exciting possibilities have arisen forpeople suffering from spinal cord injury. ES cell-derived neural cellshave been used by researchers to treat nervous system disorders inanimal models. In earlier work, researchers showed that mouse ES cellscould be stimulated to differentiate into neural cells that, whentransplanted into mice with neurological disorders, helped to restorenormal function.

However there remain obstacles in the development of such hESC-basedtreatments. Such obstacles include obtaining and maintaining adequatenumbers of undifferentiated hESCs in tissue culture and controllingtheir differentiation in order to produce specific cell types. Stem cellcultures, such as hESC cell cultures are typically seeded with a smallnumber of cells from a cell bank or stock and then amplified in theundifferentiated state until differentiation is desired for a giventherapeutic application To accomplish this, the hESC or theirdifferentiated cells are currently cultured in the presence of surfacesor media containing animal-derived components, such as feeder layers,fetal bovine serum, or MATRIGEL®. These animal-derived additions to theculture environment expose the cells to potentially harmful viruses orother infectious agents which could be transferred to patients orcompromise general culture and maintenance of the hESCs. In addition,such biological products are vulnerable to batch variation, immuneresponse and limited shelf-life.

Some steps have been taken to culture hESCs either in media or onsurfaces that are free of animal-derived components. However, theresponse of hESCs or their differentiated derivatives is difficult topredict as components of the surface or culture medium change. Yet someadvances have been made. For example, hESC-derived oligodendrocyteprogenitor cells (OPCs) have been cultured in defined serum-free medium.While such culture systems are not completely xeno-free culture systemswhen the matrices employed contain animal-derived components, such asgelatin and MATRIGEL, they do provide a step toward the eventualclinical application of hESC-derived OPCs. By way of further example,some synthetic surfaces have been identified that can supportdifferentiation of human epithelial stem cells into epithelial cells.However, the systems employed relied on serum medium for the cellculture, which still potentially causes problem as described before forall biological animal derived components. To date, a completely animalfree system employing a chemically defined medium and a syntheticsurface has not yet been identified for culturing stem cells or cellsderived from stem cells.

BRIEF SUMMARY

The present disclosure describes, inter alia, synthetic surfaces usefulin the culture of stem cell-derived OPCs in chemically defined media.

In an embodiment, a method for culturing oligodendrocyte progenitorcells is provided. The method includes depositing a suspensioncontaining the oligodendrocyte progenitor cells on a polymer materialand culturing the deposited oligodendrocyte progenitor cells in a cellculture medium. The polymer material comprises a homopolymer orcopolymer of selected one or more acrylate monomers.

In an embodiment, a culture of oligodendrocyte progenitor cells isprovided. The culture includes an article having a polymer materialdisposed on a surface. The culture further includes the oligodendrocyteprogenitor cells disposed on the polymer material and a culture mediumin which the oligodendrocyte progenitor cells are cultured. The polymermaterial comprises a homopolymer or copolymer of selected one or moreacrylate monomers.

In an embodiment, a cell culture article for culturing oligodendrocyteprogenitor cells in a chemically defined medium is provided. The articleincludes a substrate having a surface and a polymer material disposed onthe surface. The polymer material comprises a homopolymer or copolymerof selected one or more acrylate monomers.

One or more of the various embodiments presented herein provide one ormore advantages over prior surfaces for culturing stem cell-derivedOPCs. For example, the synthetic surfaces reduce potential contaminationissues associated with surfaces having components obtained from orderived from animal sources. Such surfaces may also provide for improvedshelf life compared to those surfaces with biological components. Theability to culture stem cell-derived OPCs in chemically-defined mediafurther reduces potential contamination issues. In addition, there willlikely be less batch to batch variation in the ability of the syntheticsurfaces or chemically defined media, resulting in improvedreproducibility of culture results and expectations. These and otheradvantages will be readily understood from the following detaileddescriptions when read in conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A and FIG. 1B are schematic diagrams of side views of syntheticpolymer layer coated articles.

FIGS. 2A-C are schematic diagrams of cross sections of cell culturearticles having a well. Uncoated (FIG. 2A); coated surface (FIG. 2B);and coated surface and side walls (FIG. 2C).

FIGS. 3A-C are florescence images of immunostained hES cell-derived OPCsafter replating on acrylate coating surface 123-2 (FIG. 3A) and 95-2(FIG. 3B), and positive control surface Matrigel® (FIG. 3C) between28-day and 35-day. OPCs derived from human ES cells were immunostainedfor the OPC marker, Olig1 (green) and nestin (red).

FIGS. 4A-C are fluorescence images of immunostained hES cell-derivedOPCs after replating on acrylate coated surface 123-2 (FIG. 4A), 122-3(FIG. 4B) and Matrigel® (FIG. 4C) between 28-day and 35-day as well asbetween 35-day and 42-day. OPCs derived from human ES cells wereimmunostained for the OPC marker, Olig1 (green) and nestin (red).

FIGS. 5A-F are fluorescence images of hES cell-derived OPCs on Matrigel®(FIG. 5A) and on acrylate coated surfaces 22-2 (FIG. 5B), 22-3 (FIG.5C), 133-4 (FIG. 5D), 24-10 (FIG. 5E), and 72-2 (FIG. 5F).

The drawings are not necessarily to scale Like numbers used in thefigures refer to like components, steps and the like. However, it willbe understood that the use of a number to refer to a component in agiven figure is not intended to limit the component in another figurelabeled with the same number. In addition, the use of different numbersto refer to components is not intended to indicate that the differentnumbered components cannot be the same or similar.

DETAILED DESCRIPTION

In the following detailed description, reference is made to theaccompanying drawings that form a part hereof, and in which are shown byway of illustration several specific embodiments of devices, systems andmethods. It is to be understood that other embodiments are contemplatedand may be made without departing from the scope or spirit of thepresent disclosure. The following detailed description, therefore, isnot to be taken in a limiting sense.

All scientific and technical terms used herein have meanings commonlyused in the art unless otherwise specified. The definitions providedherein are to facilitate understanding of certain terms used frequentlyherein and are not meant to limit the scope of the present disclosure.

As used in this specification and the appended claims, the singularforms “a”, “an”, and “the” encompass embodiments having pluralreferents, unless the content clearly dictates otherwise. As used inthis specification and the appended claims, the term “or” is generallyemployed in its sense including “and/or” unless the content clearlydictates otherwise.

Unless stated otherwise, ratios of compounds in a composition, such as asolution, are stated on a by volume basis.

As used herein, “have”, “having”, “include”, “including”, “comprise”,“comprising” or the like are used in their open ended sense, andgenerally mean “including, but not limited to”.

The present disclosure describes, inter alia, articles having syntheticsurfaces for culturing stem cell-derived OPCs and methods for culturingstem cell-derived OPCs on such surfaces. In some embodiments, thesynthetic surfaces are used in combination with a chemically definedmedium to culture stem cell-derived OPCs. The surfaces may be useful indifferentiating stem cells, such as hESCs, into OPCs or forproliferating such stem cell-derived OPCs.

1. Cell Culture Article

Referring to FIG. 1, a schematic diagram of article 100 for culturingcells is shown. The article 100 includes a base material substrate 10having a surface 15. A synthetic polymer coating layer 20 is disposed onthe surface 15 of the base material 10. While not shown, it will beunderstood that synthetic polymer coating 20 may be disposed on aportion of base material 10. The base material 10 may be any materialsuitable for culturing cells, including a ceramic substance, a glass, aplastic, a polymer or co-polymer, any combinations thereof, or a coatingof one material on another. Such base materials 10 include glassmaterials such as soda-lime glass, pyrex glass, vycor glass, quartzglass; silicon; plastics or polymers, including dendritic polymers, suchas poly(vinyl chloride), poly(vinyl alcohol), poly(methyl methacrylate),poly(vinyl acetate-maleic anhydride), poly(dimethylsiloxane)monomethacrylate, cyclic olefin polymers, fluorocarbon polymers,polystyrenes, polypropylene, polyethyleneimine; copolymers such aspoly(vinyl acetate-co-maleic anhydride), poly(styrene-co-maleicanhydride), poly(ethylene-co-acrylic acid) or derivatives of these orthe like.

Examples of articles 100 suitable for cell culture include single andmulti-well plates, such as 6, 12, 96, 384, and 1536 well plates, jars,petri dishes, flasks, beakers, plates, roller bottles, slides, such aschambered and multichambered culture slides, tubes, cover slips, cups,spinner bottles, perfusion chambers, bioreactors, CellSTACKs® andfermenters.

Synthetic polymer coating 20 provides a surface 25 on which cells may becultured. The synthetic polymer surface 20 includes polymerized acrylatemonomers, selected from the group of monomers provided in Table 1 below.Other materials (not shown), such as peptides, may be incorporated intoor conjugated to synthetic polymer surface to produce a biomimeticsurface.

TABLE 1 List of acrylate monomers Monomer name Monomer structureTetra(ethylene glycol) diacrylate

Glycerol dimethacrylate

Triethylene glycol dimethacrylate

1,4-Butanediol dimethacrylate

Poly(ethylene glycol) diacrylate, M_(n) ~ 258

1,6-Hexanediol diacrylate

3-Hydroxy-2,2- dimethylpropyl 3- hydroxy-2,2- dimethylpropionate

Neopentyl glycol propoxylate(1PO/OH) diacrylate

Di(ethylene glycol) diacrylate

Di(ethylene glycol) dimethacrylate

Tetra(ethylene glycol) dimethacrylate

1,6-Hexanediol propoxylate diacrylate

Glycerol 1,3- diglycerolate diacrylate

Neopentyl glycol diacrylate

Neopentyl glycol dimethacrylate

Trimethylolpropane benzoate diacrylate

Trimethylolpropane ethoxylate(1 EO/OH) methyl

Tricyclo[5.2.1.0^(2,6)]- decanedimethanol diacrylate

Neopentyl glycol ethoxylate diacrylate

Trimethylolpropane triacrylate

1,6-Hexanediol ethoxylate diacrylate M_(n) ~314

2,2,3,3,4,4,5,5 octafluoro 1,6 hexanediol diacrylate

Poly(propylene glycol) diacrylate

1,9 nonanediol diacrylate

The acrylates listed in Table 1 may be synthesized as known in the artor obtained from a commercial vendor, such as Polysciences, Inc., SigmaAldrich, Inc., and Sartomer, Inc.

As shown in FIG. 1B, an intermediate layer 30 may be disposed betweensurface 15 of base material 10 and the synthetic polymer coating 20.Intermediate layer 30 may be configured to improve binding of coating 20to substrate 10, to facilitate monomer spreading, to render portions ofthe surface 10 that are uncoated cytophobic to encourage cell growth oncoated areas, to provide a substrate compatible with a monomer orsolvent where the monomer or solvent is incompatible with the basematerial 10, to provide topographical features if desired through, forexample, patterned printing, or the like. For example, if substrate 10is a glass substrate, it may be desirable to treat a surface of theglass substrate with a silane molecule or an epoxy coating. For variouspolymer base materials 10 it may be desirable to provide an intermediatelayer 30 of polyamide, polyimide, polypropylene, polyethtylene, orpolyacrylate. While not shown, it will be understood that syntheticpolymer coating 20 may be disposed on a portion of intermediate layer30. It will be further understood that intermediate layer 30 may bedisposed on a portion of base material 10.

In various embodiments, surface 15 of base material 10 is treated,either physically or chemically, to impart a desirable property orcharacteristic to the surface 15. For example, and as discussed below,surface 15 may be corona treated or plasma treated. Examples of vacuumor atmospheric pressure plasma include radio frequency (RF) andmicrowave plasmas both primary and secondary, dielectric barrierdischarge, and corona discharge generated in molecular or mixed gasesincluding air, oxygen, nitrogen, argon, carbon dioxide, nitrous oxide,or water vapor.

Synthetic polymer coating layer 20, whether disposed on an intermediatelayer 30 or base material 10, preferably uniformly coats the underlyingsubstrate. By “uniformly coated”, it is meant that the layer 20 in agiven area, for example a surface of a well of a culture plate,completely coats the area at a thickness of about 5 nm or greater. Whilethe thickness of a uniformly coated surface may vary across the surface,there are no areas of the uniformly coated surfaces through which theunderlying layer (either intermediate layer 30 or base material 10) isexposed. Cell responses across non-uniform surfaces tend to be morevariable than cell responses across uniform surfaces.

Synthetic polymer coating layer 20 may have any desirable thickness.However, it has been found that thicker coatings, e.g. coatings ofgreater than about 10 micrometers, tend to have unevenness around theperiphery of the coating due to surface tension. In various embodiments,the thickness of the coating layer 20 is less than about 10 micrometers.For example, the thickness may be less than about 5 micrometers, lessthan about 2 micrometers, less than about 1 micrometers, less than about0.5 micrometers or less than about 0.1 micrometers.

The polymer material forming synthetic polymer layer 20 may becross-linked to any suitable degree. Higher degrees of cross-linking mayresult in reduced waste product and reduced cell toxicity.

Article 100, in numerous embodiments, is cell culture ware having awell, such as a petri dish, a multi-well plate, a flask, a beaker orother container having a well. Referring now to FIG. 2, article 100formed from base material 10 may include one or more wells 50. Well 50includes a sidewall 55 and a surface 15. Referring to FIG. 2B-C, asynthetic polymer coating 20 may be disposed on surface 15 or sidewalls55 (or, as discussed above with regard to FIG. 1 one or moreintermediate layer 30 may be disposed between surface 15 or sidewall 55and synthetic polymer coating 20) or a portion thereof.

In various embodiments, article 100 includes a uniformly coated layer 20having a surface 25 with an area greater than about 5 mm². When the areaof the surface 15 is too small, reliable cell responses may not bereadily observable because some cells, such as human embryonic stemcells, are seeded as colonies or clusters of cells (e.g., having adiameter of about 0.5 mm) and adequate surface is desirable to ensureattachment of sufficient numbers of colonies to produce a quantitativecell response. In numerous embodiments, an article 100 has a well 50having a uniformly coated surface 15, where the surface 15 has an areagreater than about 0.1 cm², greater than about 0.3 cm², greater thanabout 0.9 cm², or greater than about 1 cm².

2. Coating of Synthetic Polymer Layer

A synthetic polymer layer may be disposed on a surface of a cell culturearticle via any known or future developed process. Preferably, thesynthetic polymer layer provides a uniform layer that does notdelaminate during typical cell culture conditions. The synthetic polymersurface may be associated with the base material substrate via covalentor non-covalent interactions. Examples of non-covalent interactions thatmay associate the synthetic polymer surface with the substrate includechemical adsorption, hydrogen bonding, surface interpenetration, ionicbonding, van der Waals forces, hydrophobic interactions, dipole-dipoleinteractions, mechanical interlocking, and combinations thereof.

In various embodiments, the base material substrate surface is coatedaccording to the teachings of co-pending application Ser Ser. No.______, filed on even date herewith, naming Gehman et al. as inventors,having attorney docket number 20726, and entitled STEM CELL CULTUREARTICLE AND SCREENING, which is hereby incorporated herein by referencein its entirety for all purposes to the extent that it does not conflictwith the disclosure presented herein.

In numerous embodiments, monomers are deposited on a surface of a cellculture article and polymerized in situ. In such embodiments, the basematerial will be referred to herein as the “substrate” on which thesynthetic polymer material is deposited. Polymerization may be done insolution phase or in bulk phase.

As many of the monomers identified in Table 1 above are viscous, it maybe desirable to dilute the monomers in a suitable solvent to reduceviscosity prior to being dispensed on the surface. Reducing viscositymay allow for thinner and more uniform layers of the synthetic polymermaterial to be formed. One of skill in the art will be able to readilyselect a suitable solvent. Preferably the solvent is compatible with thematerial forming the cell culture article and the monomers. It may bedesirable to select a solvent that is non-toxic to the cells to becultured and that does not interfere with the polymerization reaction.Alternatively, or in addition, selection of a solvent that can besubstantially completely removed or removed to an extent that it isnon-toxic or no longer interferes with polymerization may be desirable.In additional embodiments, it may be desirable to select solvents whichdo not interact with the substrate. Further, it may be desirable thatthe solvent be readily removable without harsh conditions, such asvacuum or extreme heat. Volatile solvents are examples of such readilyremovable solvents. As described in co-pending application Ser. No.______, ethanol may be a particularly suitable solvent when it isdesired to remove solvent prior to polymerization.

The monomers may be diluted with solvent by any suitable amount toachieve the desired viscosity and monomer concentration. Generally themonomer compositions used according to the teachings presented hereincontain between about 0.1% to about 99% monomer. By way of example, themonomer may be diluted with an ethanol solvent to provide a compositionhaving between about 0.1% and about 50% monomer, or from about 0.1% toabout 10% monomer by volume. The monomers may be diluted with solvent sothat the polymer layer 20 achieves a desired thickness. As discussedabove, if the deposited monomers are too thick, an uneven surface mayresult. As described in further details in the Examples, uneven surfacesmay be observed when the monomer-solvent composition is deposited on asurface 15 of a well 50 at a volume of greater than about 8 microlitersper square centimeter of the surface 15. In various embodiments, themonomer-solvent compositions are deposited on a surface 15 of a well 50in a volume of about 7 microliters or less per square centimeter of thesurface 15. For example, the monomer-solvent compositions may bedeposited on a surface 15 of a well 50 in a volume of about 5microliters or less per square centimeter of the surface 15, or about 2microliters or less per square centimeter of the surface 15.

In various embodiments, article 100 includes a uniformly coated layer 20having a surface 25 with an area greater than about 5 mm². When the areaof the surface 15 is too small, reliable cell responses may not bereadily observable because some cells, such as human embryonic stemcells, are seeded as colonies or clusters of cells (e.g., having adiameter of about 0.5 mm) and adequate surface is desirable to ensureattachment of sufficient numbers of colonies to produce a quantitativecell response. In numerous embodiments, an article 100 has a well 50having a uniformly coated surface 15, where the surface 15 has an areagreater than about 0.1 cm², greater than about 0.3 cm², greater thanabout 0.9 cm², or greater than about 1 cm².

In various embodiments, synthetic polymer surface is deposited on asurface of an intermediate layer that is associated with the basematerial via covalent or non-covalent interactions, either directly orvia one or more additional intermediate layers (not shown). In suchembodiments, the intermediate layer will be referred to herein as the“substrate” onto which the synthetic polymer surface is deposited.

In various embodiments, the surface of the base material is treated. Thesurface may be treated to improve binding of the synthetic polymersurface to the base material surface, to facilitate monomer spreading onthe base material surface, or the like. Of course, the base material maybe treated for similar purposes with regard to an intermediate layer. Invarious embodiments, the surface is corona treated or vacuum plasmatreated. High surfaces energy obtainable from such treatments mayfacilitate monomer spreading and uniform coating. Examples of vacuumplasma treatment that may be employed include microwave vacuum plasmatreatments and radio frequency vacuum plasma treatments. The vacuumplasma treatments may be performed in the presence of reactive gases,such as oxygen, nitrogen, ammonia or nitrous oxide.

To form the synthetic polymer surface, one or more monomers presented inTable 1 above are polymerized. If one monomer is used, the polymer willbe referred to as a homopolymer of the monomer. If two or more differentmonomers are used, the polymer will be referred to as a copolymer of themonomers. The monomers employed may be monofunctional, difunctional, orhigher-functional. When two or more monomers are used, the ratio of themonomers may be varied. In various embodiments, two monomers are usedand the ratio, by volume of the first monomer to the second monomerranges from between about 5:95 to about 95:5. For example, the ratio ofthe first monomer to the second monomer ranges from between about 10:90to about 90:10, about 20:80 to about 80:20, from about 30:70 to about70:30. In some embodiments, the ratio of the first monomer to the secondmonomer is about 50:50, 30:70, or 10:90. It will be understood that thedegree of cross-linking of the polymer may be controlled by varying theconcentration of monomers or the ratios of difunctional orhigher-functional monomers to monofunctional monomers. Increasedconcentrations of difunctional or higher-functional monomers willincrease the degree of cross-linking in the chains.

In addition to the monomers that form the polymer layer, a compositionforming the layer may include one or more additional compounds such assurfactants, wetting agents, photoinitiators, thermal initiators,catalysts, activators, and cross-linking agents.

Any suitable polymerization initiator may be employed. One of skill inthe art will readily be able to select a suitable initiator, e.g. aradical initiator or a cationic initiator, suitable for use with themonomers listed in Table 1. In various embodiments, UV light is used togenerate free radical monomers to initiate chain polymerization.

Any suitable initiator may be used. Examples of polymerizationinitiators include organic peroxides, azo compounds, quinones, nitrosocompounds, acyl halides, hydrazones, mercapto compounds, pyryliumcompounds, imidazoles, chlorotriazines, benzoin, benzoin alkyl ethers,diketones, phenones, or mixtures thereof. Examples of suitablecommercially available, ultraviolet-activated and visiblelight-activated photoinitiators have tradenames such as IRGACURE 651,IRGACURE 184, IRGACURE 369, IRGACURE 819, DAROCUR 4265 and DAROCUR 1173commercially available from Ciba Specialty Chemicals, Tarrytown, N.Y.and LUCIRIN TPO and LUCIRIN TPO-L commercially available from BASF(Charlotte, N.C.)

A photosensitizer may also be included in a suitable initiator system.Representative photosensitizers have carbonyl groups or tertiary aminogroups or mixtures thereof. Photosensitizers having a carbonyl groupinclude benzophenone, acetophenone, benzil, benzaldehyde,o-chlorobenzaldehyde, xanthone, thioxanthone, 9,10-anthraquinone, andother aromatic ketones. Photosensitizers having tertiary amines includemethyldiethanolamine, ethyldiethanolamine, triethanolamine,phenylmethyl-ethanolamine, and dimethylaminoethylbenzoate. Commerciallyavailable photo sensitizers include QUANTICURE ITX, QUANTICURE QTX,QUANTICURE PTX, QUANTICURE EPD from Biddle Sawyer Corp.

In general, the amount of photosensitizer or photoinitiator system mayvary from about 0.01 to 10% by weight.

Examples of cationic initiators include salts of onium cations, such asarylsulfonium salts, as well as organometallic salts such as ion arenesystems.

In various embodiments where the monomers are diluted in solvent beforebeing deposited on the substrate surface, the solvent is removed priorto polymerizing. The solvent may be removed by any suitable mechanism orprocess. As described in copending application Ser. No. ______, it hasbeen found that removal of substantially all of the solvent prior tocuring, allows for better control of curing kinetics and the amount ofmonomer converted. When conversion rates of the monomers are increased,waste generation and cytotoxicity are reduced.

Whether polymerized in bulk phase (substantially solvent free) orsolvent phase, the monomers are polymerized via an appropriateinitiation mechanism. Many of such mechanisms are well known in the art.For example, temperature may be increased to activate a thermalinitiator, photoinitiators may be activated by exposure to appropriatewavelength of light, or the like. According to numerous embodiments, themonomer or monomer mixture is cured using UV light. The curingpreferably occurs under inert gas protection, such as nitrogenprotection, to prevent oxygen inhibition. Suitable UV light combinedwith gas protection may increase polymer conversion, insure coatingintegrity and reduce cytotoxicity.

The cured synthetic polymer layer may be washed with solvent one or moretimes to remove impurities such as unreacted monomers or low molecularweight polymer species. In various embodiments, the layer is washed withan ethanol solvent, e.g. 70% ethanol, greater than about 90% ethanol,greater than about 95% ethanol, or greater than about 99% ethanol.Washing with an ethanol solvent may not only serve to remove impurities,which may be cytotoxic, but also can serve to sterilize the surfaceprior to incubation with cells.

3. Incubating Cells on Synthetic Polymer Layer

Stem cell-derived OPCs may be cultured on a synthetic polymer layer, asdescribed above, according to any suitable protocol. As used herein,“stem cell derived OPC” means an OPC obtained from differentiation of astem cell. In some embodiments, the stem cells are multipotent,totipotent, or pluripotent stem cells. The stem cells may be present inan organ or tissue of a subject. In numerous embodiments, the stem cellsare embryonic stem cells, such as human embryonic stem cells. As usedherein, “OPC” or “oligodendrocyte progenitor cell” means precursor cellsto myelin-forming oligodendrocytes.

Because human embryonic stem cells (hESC) have the ability to growncontinually in culture in an undifferentiated state, the hESC for use inthis invention may be obtained from an established cell line. Examplesof human embryonic stem cell lines that have been established include,but are not limited to, H1, H7, H9, H13 or H14 (available from WiCellestablished by the University of Wisconsin) (Thompson (1998) Science282:1145); hESBGN-01, hESBGN-02, hESBGN-03 (BresaGen, Inc., Athens,Ga.); HES-1, HES-2, HES-3, HES-4, HES-5, HES-6 (from ES CellInternational, Inc., Singapore); HSF-1, HSF-6 (from University ofCalifornia at San Francisco); I 3, I 3.2, I 3.3, I 4, I 6, I 6.2, J 3, J3.2 (derived at the Technion-Israel Institute of Technology, Haifa,Israel); UCSF-1 and UCSF-2 (Genbacev et al., Fertil. Steril.83(5):1517-29, 2005); lines HUES 1-17 (Cowan et al., NEJM350(13):1353-56, 2004); and line ACT-14 (Klimanskaya et al., Lancet,365(9471):1636-41, 2005). Embryonic stem cells used in the invention mayalso be obtained directly from primary embryonic tissue. Typically thisis done using frozen in vitro fertilized eggs at the blastocyst stage,which would otherwise be discarded.

OPCs according to the invention may also be differentiated from inducedprimate pluripotent stem (iPS) cells. iPS cells refer to cells, obtainedfrom a juvenile or adult mammal, such as a human, that are geneticallymodified, e.g., by transfection with one or more appropriate vectors,such that they are reprogrammed to attain the phenotype of a pluripotentstem cell such as an hESC. Phenotypic traits attained by thesereprogrammed cells include morphology resembling stem cells isolatedfrom a blastocyst as well as surface antigen expression, gene expressionand telomerase activity resembling blastocyst derived embryonic stemcells. The iPS cells typically have the ability to differentiate into atleast one cell type from each of the primary germ layers: ectoderm,endoderm and mesoderm and thus are suitable for differentiation intoOPCs. The iPS cells, like hESC, also form teratomas when injected intoimmuno-deficient mice, e.g., SCID mice. (Takahashi et al., (2007) Cell131(5):861; Yu et al., (2007) Science 318:5858).

Stem cell derived OPCs may be obtained by any suitable methods. One wayto obtain such cells is described in Zhang et al., “Oligodendrocyteprogenitor cells derived from human embryonic stem cells expressneurotrophic factors, Stem Cells and Development, 15: 943-952 (2006),citing Nistor et al., Human embryonic stem cells differentiate intooligodendrocytes in high purity and mylenate after spinal cordtransplantation, Glia 49: 385-396 (2005). Briefly, undifferentiatedhuman embryonic stem cells, such as those derived from the H1 or H7human embryonic stem cell lines, may be cultured on MATRIGEL-coatedplates in mouse embryonic fibroblast (MEF) conditioned medium (CM)supplemented with about 8 ng/ml fibroblast growth factor-2 (FGF-2) or ina chemically defined medium, such as X-VIVO 10 from Cambrex,supplemented with about 80 ng/ml FGF-2 and 0.5 ng/ml transforming growthfactor-β1 (TGF-β1). To induce differentiation, the protocol described byNistor et al. may be employed. Briefly, the human embryonic stem cellsmay be collagenase digested, scraped, and cultured in defined mediumsupplemented with insulin, transferrin, progesterone, putrescin,selenium, triiodothyroidin and B27 for 28 days on anultra-low-attachment plate. The cells may then be cultured in thedefined medium for an additional 14 days on growth-factor reducedMATRIGEL. The cells may then be treated with FGF-2, epidermal growthfactor (EGF) and all-trans retinoic acid on specified days duringdifferentiation. Differentiation may occur over a number of days, suchas 42 days. Of course, any other suitable method may be employed.

Prior to seeding cells, the cells may be harvested and suspended in asuitable medium, such as a growth medium in which the cells are to becultured once seeded onto the surface. For example, the cells may besuspended in and cultured in serum-containing medium, a conditionedmedium, or a chemically-defined medium. As used herein,“chemically-defined medium” means cell culture media that contains nocomponents of unknown composition. Chemically defined media may, invarious embodiments, contain no proteins, hydrosylates, or peptides ofunknown composition. In some embodiments, conditioned media containspolypeptides or proteins of known composition, such as recombinantgrowth hormones. Because all components of chemically-defined media havea known chemical structure, variability in culture conditions and thuscell response can be reduced, increasing reproducibility. In addition,the possibility of contamination is reduced. Further, the ability toscale up is made easier due, at least in part, to the factors discussedabove.

The cells may be seeded at any suitable concentration. Typically, thecells are seeded at about 10,000 cells/cm² of substrate to about 500,000cells/cm². For example, cells may be seeded at about 50,000 cells/cm² ofsubstrate to about 150,000 cells/cm². However, higher and lowerconcentrations may readily be used. The incubation time and conditions,such as temperature CO₂ and O₂ levels, growth medium, and the like, willdepend on the nature of the cells being cultured and can be readilymodified. The amount of time that the cells are incubated on the surfacemay vary depending on the cell response being studied or the cellresponse desired.

Any suitable method may be used, if desired, to confirm that the stemcell derived OPCs are indeed OPCs or that the stem cells employed havesuccessfully differentiated into OPCs. For example, the presence ofcertain OPC-selective markers may be investigated. Such markers includeNestin, Oligo1, platelet derived growth factor receptor alpha (PDGFRα)and NG2. Antibodies to such markers may be used in standardimmunocytochemical or flow cytometry techniques. In addition oralternatively, cellular morphology or production of growth factorsdetectable in the medium may be evaluated. For example, cultured OPCsmay produce one or more of activin A, HGF, midkine, and TGF-β2, whichmay be detectable in the culture medium via standard assays, such asELISA.

The cultured stem cell derived OPCs may be used for any suitablepurpose, including investigational studies in culture, in animals, fordeveloping therapeutic uses, or for therapeutic purposes. One potentialtherapeutic or investigational purpose is repairing damage due to spinalcord injury.

In the following, non-limiting examples are presented, which describevarious embodiments of the articles and methods discussed above.

EXAMPLES Example 1: Identification of Acrylic Coating Surfaces Suitablefor Culturing Stem Cell Derived OPCs in a Chemically Defined Medium 1.Coating Preparation

Acrylic coating surfaces were prepared from homomonomers or copolymersof various acrylate monomers. For copolymers two different acrylatemonomers were used. A total of 24 homopolymer and 552 copolymercombinations were applied in wells. Briefly, the monomers were dilutedin ethanol, and IRGACURE 819 photoinitiator to the ratio of 1:9:0.01(monomer[volume]/ethanol[volume]/photoinitiator[weight]) to prepare theformulation. For copolymers, two different monomers were mixed with thevolume ratio of 70:30 or 30:70. In copolymer formulation, totalmonomers[volume]/ethanol[volume]/photoinitiator[weight] still remain theratio of 1:9:0.01. The formulations were placed in a well of a plasmatreated cyclic olefin copolymer 96 well plates (provided by Corning LifeScience development group) at a volume of 5 μL using BioTek PrecessionMicroplate Pipetting System. Each well received a predeterminedhomopolymer or copolymer combination, with some wells being coated withMATRIGEL as a positive control. For the wells coated with acrylatemonomers, the ethanol solvent was removed by evaporation at roomtemperature for 3 hr, which removes >99% of the ethanol. The coatingswere then cured with 13 mW/cm² pulsed (100 Hz) UV light (Xenon RC-801)for 1 min in N₂ purged box (with fused silica window). After curing, awashing step was taken. Briefly, the surface in each well of 96-wellplates was incubated with 200 μL of >99% ethanol for 1 hr followed by200 μL of water for over night to move potential extractables. Finallythe surfaces were air dried before sterilization.

2. Cell Preparation and Assays

For hESC-derived OPC, H1 hESC colonies were detached using 200 U/mlcollagenase IV and transferred to Corning ultra-low-adhesion (ULA)plates to allow the formation of embryoid bodies (EBs). To induce neuraldifferentiation, EBs were treated with epidermal growth factor (EGF),FGF-2 (fibroblast growth factor-2) and retinoic acid (RA) for 9 daysfollowing by 18 days treatment with EGF only. (Base medium: definedmedium supplemented with insulin, transferrin, progesterone, putrescin,selenium, triiodothyroidin (Sigma), and B27 (Invitrogen)).

At this point two different acrylate re-plating schedules were tested:In the first protocol, on day 28, EBs were re-plated on differentacrylic surfaces in 96-well plate or on MATRIGEL-coated wells aspositive control. Seven days later (day 35), cells were fixed with 4%PFA. In the second protocol, on day 28, EBs were re-plated on MATRIGEL,cultured for 7 days and then (day 35) re-plated on different acrylicsurfaces in 96-well plate or on MATRIGEL-coated wells as positivecontrol. Seven days later (day 42), cells were fixed with 4% PFA.

Cells from the both protocols were immunostained for OPC-specificmarkers, Nestin, Olig1, and counterstained with4′-6-Diamidino-2-phenylindole DAPI (nuclear stain).

After scanning each plate with ArrayScan, the following quantitativeanalyses were performed for the each surface: 1) TNC: total number ofcells, based on DAPI positive cell number, 2) TNO: total number of OPC,based on olig1-positive cells.

3. Results

It was found that only a small portion of the tested surfaces supportedattachment and cell outgrowth from EBs in chemically defined medium.FIG. 3 are fluorescence images of immunostained hES cell-derived OPCafter re-plating on selected acrylate coated surfaces and positivecontrol Matrigel surface between 28-day and 35-day for differentiation.FIG. 4 are fluorescence images of immunostained hES cell-derived OPCafter two re-platings on selected acrylate coated surfaces and positivecontrol Matrigel surface between 28-day and 35-day, as well as between35-day and 42-days for differentiation. The immunostaining images showedthat differentiated cells expressed the OPC markers on some acrylatecoating surfaces. The staining of the cells grown on the acrylatesurfaces was similar to the staining observed in the cells grown on theMartigel control surface. Examples of the coating surfaces whichsupported differentiated human OPCs in chemically defined medium arelisted in Table 2, where the volume ratio of monomer (1) to monomer (2)is 70:30.

TABLE 2 Example compositions of acrylic polymers which support theculture of hES cell derived OPCs in chemically defined medium. PolymerID Monomer (1) Monomer (2) 95-1 Tetra(ethylene glycol) diacrylate 27-1Tetra(ethylene glycol) diacrylate Neopentyl glycol ethoxylate diacrylate123-1  Glycerol dimethacrylate Tetra(ethylene glycol) diacrylate 123-2 Glycerol dimethacrylate Tri(ethylene glycol) dimethacrylate 22-1Glycerol dimethacrylate Di(ethylene glycol) dimethacrylate 22-2 Glyceroldimethacrylate Tetraethylene glycol dimethacrylate 22-3 Glyceroldimethacrylate 1,6-Hexanediol propoxylate diacrylate  24-10 Glyceroldimethacrylate 1,6-Hexanediol ethoxylate diacrylate 28-2 Tri(ethyleneglycol) Trimethylolpropane triacrylate dimethacrylate  28-101,4-Butanediol dimethacrylate 1,9 nonanediol diacrylate 36-41,6-Hexanediol diacrylate Tricyclo[5.2.1.0^(2,6)]decanedimethanoldiacrylate 36-5 1,6-Hexanediol diacrylate 1,6-Hexanediol ethoxylatediacrylate 36-6 1,6-Hexanediol diacrylate Neopentyl glycol ethoxylatediacrylate 39-6 Neopentyl glycol propoxylate Neopentyl glycol ethoxylatediacrylate (1PO/OH) diacrylate 133-4  Di(ethylene glycol) Neopentylglycol diacrylate dimethacrylate 47-4 Di(ethylene glycol)Tricyclo[5.2.1.0^(2,6)]decanedimethanol dimethacrylate diacrylate 41-9Tetra(ethylene glycol) 1,4-Butanediol dimethacrylate dimethacrylate 50-6Tetra(ethylene glycol) Neopentyl glycol ethoxylate diacrylatedimethacrylate 51-1 1,6-Hexanediol propoxylate Neopentyl glycolethoxylate diacrylate diacrylate 49-4 Neopentyl glycol diacrylateTricyclo[5.2.1.0^(2,6)]decanedimethanol diacrylate 49-5 Neopentyl glycoldiacrylate 1,6-Hexanediol ethoxylate diacrylate 52-4 Neopentyl glycoldiacrylate Poly(propylene glycol) diacrylate 63-3 Trimethylolpropaneethoxylate (1 2,2,3,3,4,4,5,5 octafluoro 1,6 EO/OH) methyl diacrylatehexanediol diacrylate 71-1 Neopentyl glycol ethoxylate Di(ethyleneglycol) dimethacrylate diacrylate 65-9 Trimethylolpropane triacrylate1,4-Butanediol dimethacrylate 71-6 Trimethylolpropane triacrylateDi(ethylene glycol) dimethacrylate  71-10 Trimethylolpropane triacrylateNeopentyl glycol diacrylate 74-6 Trimethylolpropane triacrylateNeopentyl glycol dimethacrylate 72-2 2,2,3,3,4,4,5,5 octafluoro 1,6Tetra(ethylene glycol) dimethacrylate hexanediol diacrylate 72-52,2,3,3,4,4,5,5 octafluoro 1,6 Neopentyl glycol diacrylate hexanedioldiacrylate 72-9 Poly(propylene glycol) Glycerol 1,3-diglycerolatediacrylate diacrylate

FIGS. 5A-F show images taken from micrographs of hESC derived OPCsgrowing on Matrigel™ as a positive control and selected embodiments ofsurfaces of the present invention; 22-2 (B), 22-3 (C), 133-4 (D), 24-10(E), and 72-2 (F). FIGS. 5A-F show nuclear staining on hESC derived OPCson Matrigel or the above-referenced embodiments of surfaces of thepresent invention stained with Hoecst nuclear stain. FIGS. 5A-Fillustrate that embodiments of surfaces of the present invention providesuitable surfaces to support adhesion and growth of hESC derived OPCs inchemically defined medium. For other tested homopolymers and copolymercombinations, that is those combinations that are not listed in Table 2above, no EB attachment to the surface or cell outgrows from the EBs wasobserved.

Thus, embodiments of SYNTHETIC SURFACES FOR CULTURING STEM CELL DERIVEDOLIGODENDROCYTE PROGENITOR CELLS are disclosed. One skilled in the artwill appreciate that the arrays, compositions, kits and methodsdescribed herein can be practiced with embodiments other than thosedisclosed. The disclosed embodiments are presented for purposes ofillustration and not limitation.

1-13. (canceled)
 14. A method for culturing pluripotent stemcell-derived oligodendrocyte progenitor cells, comprising depositing asuspension comprising the pluripotent stem cell-derived oligodendrocyteprogenitor cells on a polymer material, and culturing the depositedpluripotent stem cell-derived oligodendrocyte progenitor cells in a cellculture medium, wherein the polymer material comprises a diacrylate or adimethacrylate.
 15. The method of claim 14, wherein the dimethacrylateis glycerol dimethacrylate.
 16. The method of claim 14, wherein thediacrylate is 1,6-hexanediol diacrylate.
 17. The method of claim 14,wherein the diacrylate is tetra(ethylene glycol)diacrylate.
 18. Themethod of claim 14, wherein the polymer material comprises about 70%glycerol dimethacrylate.
 19. The method of claim 14, wherein thepluripotent stem cell-derived oligodendrocyte progenitor cells are humancells.
 20. The method of claim 14, wherein the pluripotent stemcell-derived oligodendrocyte progenitor cells are human embryonic stemcell-derived oligodendrocyte progenitor cells.
 21. The method of claim14, wherein the cell culture medium is a chemically defined medium.